We investigated the molecular mechanisms governing the low level expressed delta-globin gene, which could facilitate the therapeutic manipulation of hemoglobin A2 (HbA2) synthesis. Previously, we demonstrated that restoration of the CCAAT sequence at -70bp and/or insertion of a consensus EKLF binding site at -85bp position in a delta-promoter linked luciferase reporter system resulted in significantly increased activity in both K562 and MEL cells, as well as human primary adult erythroid cells (hAEC). Our in vitro results therefore implicate the mutated CCAAT box (CCAAC) and the lack of an EKLF binding site in the promoter at least partially responsible for the low level delta-globin gene expression in adult erythroid cells at transcriptional level. We also generated transgenic mice with EKLF binding site insertion in delta promoter sequence within the constructs containing locus control region and human beta- and delta-globin genes. We have developed a new assay with real-time PCR to quantitate the delta-globin expression level specifically. Studies are underway to evaluate the delta globin chain synthesis in these transgenic mice. In addition, we constructed five in-frame chimeric transcription molecules containing a transactivation domain from EKLF linked with two zinc-fingers from EKLF and one zinc-finger from GATA-1. One of the chimeric constructs, cotransfected with delta-luciferase reporter construct into K562 cells, increased the reporter activity by 3-4 fold. We are developing highly quantitative methods to assay the binding affinity of the chimeric proteins with different combination of linker sequences and identify the specific fusion protein that can target the defective delta-globin promoter. This novel transcription factor will be tested for targeting delta globin gene in cell lines, primary cells as well as transgenic mice.